Protocol for determining protein cysteine thiol redox status using western blot analysis

Pant, Bikram Datt; Oh, Sunhee; Mysore, Kirankumar S.

doi:10.1016/j.xpro.2021.100566

Cited by 5 publications

(3 citation statements)

References 11 publications

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“…Conserved cysteine residues are related to catalytic processes, folding, and metal binding, and also regulation of protein activity through its reversible and interconvertible oxidations that include S-sulfenylation, S-nitrosylation, Spersulfidation, and S-glutathionylation [10]. To determine if Cys127 in PepMV CP was oxidized in vivo, we performed thiol alkylation reactions under non-reducing conditions with methyl-polyethylene glycol-maleimide (MM(PEG) 24 ) [35] on total protein extracts from N. benthamiana leaves expressing CP WT , CP C127S or PepCP WT . In this assay, reduced cysteines are alkylated with MM(PEG) 24 , increasing protein mass by 1.24 kDa per each single conjugation; pre-oxidized (ox) and pre-reduced (red) protein extracts were included as controls.…”

Section: Resultsmentioning

confidence: 99%

“…An asterisk indicates statistically significant differences between groups, as computed by a one-way ANOVA (F 1,6 = 148.61, P = 0.00). (D) Western blot of protein extracts from N. benthamiana leaves expressing CP WT or CP C127S incubated with methyl-polyethylene glycol-maleimide (MM(PEG) 24 ) as described by Pant et al (2021). Reduced cysteines are alkylated with MM(PEG) 24 and one conjugation increases the protein mass by 1.24 kDa.…”

Section: Resultsmentioning

confidence: 99%

“…The CP redox assay was conducted as described by Pant et al (2021). Disks from N. benthamiana leaves agroinfiltrated with pGWB2-CP or pGWB2-CP C127S at 4 dpa were used as the starting plant material.…”

Section: Cp Redox Assaymentioning

confidence: 99%

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A regulatory role for the redox status of the pepino mosaic virus coat protein

Méndez-López,

Aranda

2023

PLoS Pathog

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Cysteine oxidations play important regulatory roles during animal virus infections. Despite the importance of redox modifications during plant infections, no plant virus protein has yet been shown to be regulated by cysteine oxidation. The potexvirus pepino mosaic virus (PepMV) is pandemic in tomato crops. Previously we modeled the structure of the PepMV particle and coat protein (CP) by cryo-electron microscopy and identified critical residues of the CP RNA-binding pocket that interact with the viral RNA during particle formation and viral cell-to-cell movement. The PepMV CP has a single cysteine residue (Cys127) central to its RNA binding pocket, which is highly conserved. Here we show that the Cys127Ser replacement diminishes PepMV fitness, and that PepMV CPWT is oxidized in vivo while CPC127S is not. We also show that Cys127 gets spontaneously glutathionylated in vitro, and that S-glutathionylation blocks in vitro the formation of virion-like particles (VLPs). VLPs longer than 200 nm could be formed after in planta CPC127S overexpression, while very short and dispersed VLPs were observed after CPWT overexpression. Our results strongly suggest that the CP redox status regulates CP functions via cysteine oxidation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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A regulatory role for the redox status of the pepino mosaic virus coat protein

Méndez-López,

Aranda

2023

PLoS Pathog

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Solution Structure and Acid‐Base Properties of Reduced α‐Conotoxin MI

Faragó

Mirzahosseini

Horváth

et al. 2021

Chemistry & Biodiversity

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The reduced derivative of α‐conotoxin MI, a 14 amino acid peptide is characterized by NMR‐pH titrations and molecular dynamics simulations to determine the protonation constants of the nine basic moieties, including four cysteine thiolates, and the charge‐dependent structural properties. The peptide conformation at various protonation states was determined. The results show that the disulfide motifs in the native globular α‐conotoxin MI occur between those cysteine moieties that exhibit the most similar thiolate basicities. Since the basicity of thiolates correlates to its redox potential, this phenomenon can be explained by the higher reactivity of the two thiolates with higher basicities. The folding of the oxidized peptide is further facilitated by the loop‐like structure of the reduced form, which brings the thiolate groups into sufficient proximity. The 9 group‐specific protonation constants and the related, charge‐dependent, species‐specific peptide structures are presented.

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